Abstract
Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen is relatively costly and the requirement for low temperatures during shipping is inconvenient. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. Thus, the glass transition temperature is a key parameter for design of cell desiccation procedures. In this study, we have investigated the effects of moisture content on the glass transition temperature (Tg) of mixtures of sugars (trehalose or raffinose), polymers (polyvinylpyrrolidone or Ficoll), penetrating cryoprotectants (ethylene glycol, propylene glycol, or dimethyl sulfoxide), and phosphate buffered saline (PBS) solutes. Aqueous solutions were dried to different moisture contents by equilibration with saturated salt solutions, or by baking at 95°C. The glass transition temperatures of the dehydrated samples were then measured by differential scanning calorimetry. As expected, Tg increased with decreasing moisture content. For example, in a desiccation medium containing 0.1 M trehalose in PBS, Tg ranged from about 360 K for a completely dry sample to about 220 K at a water mass fraction of 0.4. Addition of polymers to the solutions increased Tg, while addition of penetrating cryoprotectants decreased Tg. Our results provide insight into the relationship between relative humidity, moisture content and glass transition temperature for cell desiccation solutions containing sugars, polymers and penetrating cryoprotectants.
Highlights
Maintaining the viability mammalian cells during long term storage is important in many areas, including embryo preservation for breeding of domestic animals [1], species conservation [2] and assisted human reproduction [3], as well as preservation of cell-based therapeutic products such as stem cells [4]
When binary trehalose-water mixtures were dried from an aqueous solution, the moisture content observed after equilibration was about 0.104 at a relative humidity of 54% and dropped slightly to 0.095 at a relative humidity of 33%
When aqueous trehalose solutions were freeze-dried before equilibration in controlled relative humidity environments, much lower moisture contents were observed in the samples
Summary
Maintaining the viability mammalian cells during long term storage is important in many areas, including embryo preservation for breeding of domestic animals [1], species conservation [2] and assisted human reproduction [3], as well as preservation of cell-based therapeutic products such as stem cells [4]. The requirement for storage in liquid nitrogen increases costs and makes it more challenging to ship the cells from the storage facility to the end user To address these drawbacks, several researchers have recently begun to investigate the potential for cell desiccation to enable storage at higher temperatures [5,6,7,8,9,10,11]. In contrast to mammalian cells, several other organisms (including yeast, tardigrades and even larger organisms such as resurrection plants) can survive nearly complete dehydration [13] These organisms have developed adaptations that provide protection at low water content, most notably the accumulation of sugars such as trehalose and sucrose [13]. Another key mechanism of desiccation protection is the formation of aqueous glass, which embeds cellular components in a noncrystalline solid matrix and slows down degradative chemical reactions [14]
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